Summary: The current tuberculosis vaccine, BCG, was generated by attenuating a mycobacterial strain (Mycobacterium bovis) that is related but not identical to M. tuberculosis. It is believed that an attentauted mutant of M. tb with its full complement of tuberculosis antigens would provide more protection than the modestly active BCG vaccine. To evaluate the potential effectiveness of attentuated M. Tb strains as Tb vaccines, virulent M. tb H37Rv has been attenuated by a targeted mutagenesis. The resulting gene deletion strains have been injected into C57BL/6 mice to assess their survival in the lung and spleen as a measure of residual virulence and potential immunogenicity. To assess the protection afforded by the attenuated strains, the mice have been challenged 6-12 weeks later with a small aerogenic inoculum of M. tb Erdman and the growth in the lung was compared with that of a group of BCG controls. In collaboration with Dr. William Jacobs of the Albert Einstein University, we have extensively evaluated 7 deletion mutants of M. Tb. Four strains have deletions in genes - Leu, Lys, NAD, and Pan - involved with intracellular metabolism. The RD1 mutant contains a large deletion in the RD1 region of the TB genome; this genetic locus contains important TB virulence determinants. Additionally, we have recently studied two muliple gene deletion strains of M. tuberculosis - the RD1pan and lyspan mutants. A summary of the results generated in these studies is provided below. 1. The auxotrophic H37RvLeu- mutant induced high levels of anti-tuberculous resistance but virulence tests carried out on this auxotroph indicated an unacceptable level of reversion to the wild type and further studies with this auxotroph were discontinued. 2. Tests with lysine auxotrophs of BCG Pasteur and M. tb H37Rv have been carried out in C57BL/6 mice. Both auxotrophs failed to multiply in vitro in the absence of an extrinsic source of lysine and were unable to grow or survive in intravenously vaccinated mice. They were unable to induce detectable anti-tuberculous resistance against an aerogenic Erdman challenge administered 3 months later. However, mice which received 2 or 3 doses of the lysine auxotroph expressed enhanced resistance to challenge, approaching that observed in the BCG vaccinated controls. 3. The NAD mutant persists for many months in C57BL/6 mice. Importantly, the challenge studies have indicated that a single dose of this strain is more protective than BCG. However, our virulence studies have indicated that this strain is not fully attentuated and hence would be unacceptable for clinical use. Because the NAD strain is persistent and immunogenic, an additional gene is being deleted from this strain to reduce its virulence. We anticipate that a NAD double KO strain will be sufficiently immunogenic and less toxic and a potential vaccine candidate. 4. The PAN mutant has a deletion in a gene responsible for the production of pantothenic acid. This mutant is only modestly persistent in our mouse model. In fact, when delivered subcutanously only a few organisms are detected in the spleens and lungs at 1 and 2 months post infection. Because of this modest persistence, the toxicity of this strain is limited. Surprisingly, the protective response induced by two subcutaneously injections of this mutant is equivalent to the BCG response. Thus, the PAN mutant is a viable vaccine candidate because of its low toxicity and substantial immongenicity. 5. The RD1 mutant carries a large deletion in a region of the TB genome encoding potential virulence determinants. This mutant persists in mice for months and protects as well as BCG against an aerogenic challenge. However, studies in immune deficient mice suggest that the RD1 mutant is not fully attenuated. 6. The RD1pan muliple gene deletion strain may be a viable vaccine candidate. This strain persists in mice at a low level for several months and is as protective as BCG in our mouse model. Furthermore, studies in scid mice show that the RD1pan mutant is highly attenuated. 7. Our preliminary studies with the Lyspan multiple deletion strain indicates that it is somewhat persistent, immunogenic, and highly attenuated.